Device for solid phase extraction and method for purifying samples prior to analysis

ABSTRACT

A solid phase extraction (SPE) device having a reservoir with an opening; a well comprising an internally tapered wall, the well having a wider interior diameter at an end closest to the opening than at an exit spout; a first filter within the well; a bed of sorbent particles within the well below the first filter; and a second filter having a diameter smaller than the first filter within the well below the bed of sorbent particles and above the exit spout is provided. A method of performing SPE using the device is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS:

This application is a divisional of U.S. application Ser. No.10/785,754, filed Feb. 24, 2004 and U.S. Ser. No. 10/100,762 filed Mar.19, 2002, now U.S. Pat. No. 6,723,236. The contents of theaforementioned application are hereby expressly incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

Solid phase extraction (SPE) is a chromatographic technique forpreparing samples prior to performing quantitative chemical analysis,for example, via high performance liquid chromatography (HPLC), or gaschromatography (GC). The goal of SPE is to isolate target analytes froma complex sample matrix containing unwanted interferences, which wouldhave a negative effect on the ability to perform quantitative analysis.The isolated target analytes are recovered in a solution that iscompatible with quantitative analysis. This final solution containingthe target compound can be directly used for analysis or evaporated andreconstituted in another solution of a lesser volume for the purpose offurther concentrating the target compound, making it more amenable todetection and measurement.

Depending on the type of analysis to be performed, and detection methodused, SPE may be tailored to remove specific interferences. Analysis ofbiological samples such as plasma and urine using high performanceliquid chromatography (HPLC) generally requires SPE prior to analysisboth to remove insoluble matter and soluble interferences, and also topre-concentrate target compounds for enhanced detection sensitivity.Many sample matrices encountered in bio-separations contain buffers,salts, or surfactants, which can be particularly troublesome when massspectrometer based detection is used. SPE can also be used to perform asimple fractionation of a sample based on differences in the chemicalstructure of the component parts, thereby reducing the complexity of thesample to be analyzed.

Devices designed for SPE typically include a chromatographic sorbentwhich allows the user to preferentially retain sample components. Once asample is loaded onto the sorbent, a series of tailored washing andelution fluids are passed through the device to separate interferencesfrom target sample components, and then to collect the target samplecomponents for further analysis. SPE devices usually include a sampleholding reservoir, a means for containing the sorbent, and a fluidconduit, or spout for directing the fluids exiting the device intosuitable collection containers. The SPE device may be in a single wellformat, which is convenient and cost effective for preparing a smallnumber of samples, or a multi-well format, which is well suited forpreparing large numbers of samples in parallel. Multi-well formats arecommonly used with robotic fluid dispensing systems. Typical multi-wellformats include 48-, 96-, and 384-well standard plate formats. Fluidsare usually forced through the SPE device and into the collectioncontainers, either by drawing a vacuum across the device with aspecially designed vacuum manifold, or by using centrifugal orgravitational force. Centrifugal force is generated by placing the SPEdevice, together with a suitable collection tray, into a centrifugespecifically designed for the intended purpose.

Various means have been used to contain chromatographic sorbents withinSPE devices. A common method, described in U.S. Pat. No. 4,211,658,utilizes two porous filters, with chromatographic sorbent containedbetween the filters. In this design, the SPE device is essentially asmall chromatographic column containing a packed bed of sorbent. Avariation of this design is described in U.S. Pat. No. 5,395,521, wherethe porous filter elements are spherical in shape. In U.S. Pat. No.4,810,381, the chromatographic sorbent is immobilized within a thinporous membrane structure. In EP Application No. 1110610A1 a method isdescribed for securing these filters within the SPE device by means of asealing ring pressed around the periphery of the membrane disc. In U.S.Pat. No. 5,486,410 a fibrous structure containing immobilized functionalmaterials is described. In U.S. Pat. No. 5,906,796 an extraction plateis described where glass fiber discs containing chromatographic sorbentare press fit into each well of the SPE device.

A number of chromatographic sorbents can be used depending on the natureof the sample matrix and target compounds. A common example is to useporous silica that has been surface derivatized with octydecyl (C₁₈) oroctyl (C₈) functional groups. Porous particles that are based on organicpolymers are also widely used. One such type, which is particularly wellsuited for SPE due to its high loading capacity and unique retentionproperties, is described in U.S. Pat. No. 6,254,780.

Typical SPE methods contain a sequence of steps, each with a specificpurpose. The first step, referred to as the “conditioning” step,prepares the device for receiving the sample. For reversed-phase SPE,the conditioning step involves first flushing the SPE device with anorganic solvent such as methanol or acetonitrile, which acts to wet thesurfaces of both the device and the sorbent, and also rinses anyresidual contaminants from the device. This initial rinse is generallyfollowed with a highly aqueous solvent rinse, often containing pHbuffers or other modifiers, which will prepare the chromatographicsorbent to preferentially retain the target sample components. Onceconditioned, the SPE device is ready to receive the sample.

The second step, referred to as the “loading” step, involves passing thesample through the device. During loading, the sample components, alongwith many interferences are adsorbed onto the chromatographic sorbent.Once loading is complete, a “washing” step is used to rinse awayinterfering sample components, while allowing the target compounds toremain retained on the sorbent. The washing step is then followed by an“elution” step, which typically uses a fluid containing a highpercentage of an organic solvent, such as methanol or acetonitrile. Theelution solvent is chosen to effectively release the target compoundsfrom the chromatographic sorbent, and into a suitable sample container.

In many cases, elution with high concentrations of organic solventrequires that further steps be taken before analysis. In the case ofchromatographic analysis (HPLC), it is highly desirable for samples tobe dissolved in an aqueous-organic mixture rather than a pure organicsolvent, such as methanol or acetonitrile. For this reason, SPE sampleseluted in pure acetonitrile or methanol are usually evaporated todryness (“drydown”), and then reconstituted in a more aqueous mixture(“reconstitution”) before being injected into an HPLC system. Theseadditional steps not only take time and effort, but can also lead toloss of valuable sample, either through target analyte loss ontocollection container surfaces during drydown, or due to target analyteevaporative losses or difficulties encountered when trying tore-dissolve the dried sample in the higher percent aqueous fluid.

It can be seen then, that it is advantageous for an SPE device to have ahigh capacity for retaining target compounds of a wide range ofchromatographic polarities, to be capable of maintaining target compoundretention as sample interferences are washed to waste, and then toprovide the capability to elute target compounds in as small an elutionvolume as possible, thereby maximizing the degree of target compoundconcentration obtained during SPE.

The ability to elute in very small volumes of solvent has the addedbenefit of minimizing the amount of time required to evaporate andreconstitute the sample before proceeding with analysis if furtherconcentration or solvent exchange is required. If elution volume can bekept very low, then drydown and reconstitution can be entirelyeliminated.

Traditional SPE device designs have attempted to address these issues,each with a limited measure of success. Packed bed devices utilizepacked beds of sorbent particles contained between porous filter discsthat are press fit into the SPE device. The capture efficiency of theresulting packed beds is typically quite good, especially if the sorbentproperties are carefully chosen. One drawback with conventional packedbed devices is that the void volume contained within the porous filtersand packed bed requires that relatively large elution volumes be used tocompletely elute the target compounds. Typical elution volumes requiredto fully elute target compounds from a packed bed type SPE device fallin the range of 200-5000 μL, depending on the size of the sorbent bed.

Membrane based designs attempt to address this issue by embeddingsorbent particles within a fluorocarbon based membrane, which are thenplaced into the SPE device. A small mass of sorbent particles isembedded into a thin membrane structure with a wide cross sectionalarea. Since the membrane does not require retaining filters, the volumeassociated with the two porous filters is eliminated. This approachreduces the total volume contained within the device, and therefore thevolume of solvent required for elution. A typical elution volumerequired to fully elute target compounds from a particle in membrane SPEdevice fall in the range of 75-500 μL. Designs of this type havedrawbacks in other areas however. The sorbent particles are less denselypacked within the membrane structure than within a packed bed, leadingto poorer capture efficiency, and a greater chance that target compoundswill break through the device without being well retained. In addition,the flow properties of the membrane can be highly variable, due to thepoor wetting characteristics of the fluorocarbon based membrane whenusing highly aqueous fluids.

In U.S. Pat. No. 5,906,796 a design is described in which glass fiberbased extraction discs containing chromatographic particles are pressfit into each well of the SPE device. Like the membrane designs, thisapproach immobilizes the sorbent particles in a thin sheet, therebyminimizing device void volume and required elution volumes. Typicalvolumes required to fully elute target compounds from an SPE device suchas this fall in the range of 75-500 μL, which is comparable toparticle-in-membrane devices. The sorbent particles are even lessdensely packed than with membranes however, so sample breakthrough tendsto be higher than with either membrane or packed bed devices, andsorbent particles can often break free from the fibrous matrix andcontaminate the collected sample solution. One advantage over membranedevices is that flow problems due to wetting issues are generally lesscommon due to the more open structure of the glass fiber disc. Onedisadvantage of this particle embedded glass fiber disk is that itcontains silanol groups that interact with basic compounds. Thisrequires the use of more complex elution solvents, for example, theaddition of 2% base or acid to the elution solvent, to maintain the75-500 μL elution volumes.

It can be seen then, that the lower elution volume capability achievedwith both the membrane and glass fiber approaches is at the expense oftarget compound breakthrough during loading and/or poor recovery fornon-polar compounds. Although the volume of fluid needed to effectivelyelute samples from the membrane and glass fiber formats is reduced toapproximately one half of the volume required with conventional packedbed based devices, dry-down and reconstitution steps are still requiredbefore samples can be further analyzed by HPLC.

SUMMARY OF THE INVENTION

The present invention relates to an improved SPE device which has beenspecifically designed to contain a small packed bed of chromatographicsorbent such that the bed provides for highly efficient retention oftarget compounds, while the volume contained within the sorbent bed issufficiently small as to allow for efficient elution of sample compoundsin a minimal elution volume. Specifically, the solid phase extractiondevice of the present invention comprises a reservoir with an opening; awell comprising an internally tapered wall, the well having a widerinterior diameter at an end closest to the opening than at an exitspout; a first filter within the well; a bed of sorbent particles withinthe well below the first filter; and a second filter having a smallerdiameter than the first filter within the well below the bed of sorbentparticles and above the exit spout.

The present invention provides a large bed height to top bed diameterratio using a significantly smaller sorbent mass than is present incurrent state of the art devices. The large bed height to bed diameterratio enhances the retention of target compounds and helps to preventbreakthrough of these compounds during the load and wash steps. In SPEthe first filter and the top of the sorbent bed acts like a depth filterin removing insoluble sample components. The larger diameter for theupper portion of the bed and larger diameter first filter allows thedevice to draw through larger sample volumes than could be drawn througha device having an upper bed diameter the same as the lower bed diameterbefore obstructions will occur. The smaller second filter increases thebed height to bed diameter ratio for a given mass of sorbent whilereducing the hold up volume of the device which minimizes requiredelution volumes.

Moreover, the present invention provides for conically shaped packedbeds contained between spherical filters which enhance the performanceof solid phase extraction devices by allowing target compounds to beboth efficiently retained and eluted. The larger first spherical filterprovides a surface area that is approximately two times the area of anequivalently sized cylindrical filter. For example, surface area of thetop half of a sphere (π/2×d²) of a diameter of 0.1″ is equal to thesurface area of the top of a disk of diameter 0.14″ (π/4×d²). Thesmaller second filter helps to minimize the amount of sorbent needed tocreate a bed length that will be free of adverse imperfections.

The present invention enables the retention of target compounds with awide range of chromatographic polarity with elution in volumes that aremuch reduced from the current state of the art for solid phaseextraction. This reduction in elution volume provides a solutioncontaining the target compounds that can be diluted with an aqueoussolution while still maintaining the high sample concentrations requiredfor analysis.

According to another aspect, the present invention provides an enhancedmethod of performing solid phase extraction, where the volume of elutionsolvent is sufficiently small so as to eliminate the need for anevaporation step. The method involves elution of the target compounds ina minimal volume of organic solvent, typically 10-40 μL, which is thendiluted with a highly aqueous fluid to form an aqueous organic samplemixture. This mixture is suitable for direct analysis by HPLC, therebyeliminating the time, expense, and potential sample losses associatedwith evaporation and reconstitution steps, while still maintaining ahigh degree of target compound(s) concentration.

Specifically, the inventive method comprises the steps of providing theabove-mentioned SPE device, and isolating target substances frominterfering components in a sample medium, wherein the target substancesare substantially eluted in less than 50 μL volume.

In one embodiment of the present invention, the isolating step of thepresent invention preferably includes conditioning the SPE device withan organic solvent; equilibrating the SPE device with an aqueoussolution; adding a prepared sample containing the target substances andinterfering components to the SPE device; washing the SPE device with anaqueous solution to remove interfering components; and eluting theadsorbed target substances.

In a preferred embodiment of this enhanced method of the presentinvention, the aqueous diluent is added directly through the SPE device,while still on the processing station used to perform the SPE fluidtransfers. In this way, residual elution solvent is swept through thedevice into the collection container, where it is diluted by the aqueousfluid and mixed by the gentle air stream that is drawn through the wellat the end of the transfer. This approach has the advantage ofeliminating the need for a separate pipetting operation to perform thedilution step.

SUMMARY OF THE DRAWINGS

FIG. 1 is an illustration of a single well embodiment of the presentinvention, where the internal well geometry is a simple tapered shape,containing two spherical filter elements of different sizes, a bed ofsorbent particles contained between the filters, an exit spoutdownstream from the smaller filter, and a fluid reservoir upstream fromthe larger porous filter.

FIG. 2 is an illustration of a single well embodiment of the presentinvention where the internal tapered well geometry is segmented,providing an exit spout downstream from the smaller porous filter, alower tapered section containing both the smaller porous filter and abed of sorbent particles, an upper tapered section which contains thelarger porous filter upstream from the sorbent bed, and a transitionsection leading to an upper fluid reservoir.

FIG. 3 is an illustration of a multi-well SPE device where each well ofthe device contains the single well device geometry of FIG. 2.

FIG. 4 is a graph illustrating the effect of hold-up volume on recoveryin 25 μL elution volumes.

FIG. 5 is an illustration of a trapezoidal exit spout.

FIG. 6 is an illustration of a semi-circular exit spout of the device.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 shows an SPE device of packed bed design where the device hasbeen optimized for high capture efficiency, while requiring minimalelution volume. The device (11) contains a small bed of sorbentparticles (12), contained between two porous filter elements ofdifferent sizes (13 & 14) within a tapered internal well geometry (15),such that the porous filter (13) positioned downstream from the sorbentbed (12) is smaller than the porous filter (14) positioned upstream fromthe sorbent bed. The device also includes a reservoir section (16)located upstream from the larger porous filter (14) and an exit spout(17) located downstream from the smaller porous filter (13). The spoutdirects fluids exiting the device into a suitable collection container(not shown).

Porous filters (13 & 14) may be of any material suitable for retainingthe sorbent particles. In a preferred embodiment, porous filters (13 &14) are made from sintered polyethylene material. FIG. 1 represents asingle well version of the device, although it will be obvious to oneskilled in the art, that the device may also be configured as part of amulti-well SPE device.

In this preferred embodiment of the SPE device of the present invention,the porous filters that contain the sorbent particles are spherical inshape, and the sorbent bed is configured in a tapered geometry, with thedownstream porous spherical filter being smaller than the upstreamporous spherical filter.

The sorbent particles employed in the device include any particulatematter that is capable of having at least one substance, either targetor interfering, adhered thereto. Illustrative examples of sorbentparticles that may be employed in the present invention include, but arenot limited to: ion exchange sorbents, reversed phase sorbents, andnormal phase sorbents. More particularly, the sorbent particles may bean inorganic material such as SiO₂ or an organic polymeric material suchas poly(divinylbenzene). In some embodiments of the present invention,the sorbent particles may be treated with an organic functional groupsuch as a C₂-C₂₂, preferably C₈-C₁₈ functional group. One skilled in theart will find it obvious that the size, shape, surface area, and porevolume of the sorbent particles, may all be modified to suit specificapplications without departing from the scope of the invention.

The tapered internal device geometry acts to provide an upstream firstporous filter having a large filtration area for capturing foreignsample particulates prior to them reaching the sorbent bed, and asmaller downstream filter, while allowing minimal internal void volumebetween the sorbent bed and the first filter. The effective filtrationarea of the spherical filter is based on the surface of the exposedhemispherical section of the filter, which is larger than the area of aflat disc filter of equal diameter by a factor of 2.

The spherical filters are easy to handle during assembly and require nospecial insertion tooling. Moreover, the spherical filters self-alignwhen placed into a tapered well cavity, and seal against the cavity walleasily without the need for close dimensional tolerances between thespherical filters and the internal surface of the well. The tapered welldesign also allows for a range of sorbent masses within the same SPEdevice, thereby providing flexibility to tailor the device for differentapplications. This is accomplished by simply changing the diameter ofthe spherical porous filters, thereby positioning the filters and packedsorbent bed either higher or lower within the tapered device withouthaving to alter the well cavity.

The tapered well geometry differs from conventional cylindrical designs,since it results in a sorbent bed shape that has considerably lesstendency to form undesirable flow channels, thereby preventing samplecomponents bypassing the bed without adequately contacting the sorbentparticles. Fluids passing through the sorbent bed during theconditioning and loading steps act to consolidate the tapered packedbed, resulting in a consistently formed bed structure. This results inefficient contact between the sample and the sorbent bed, less chancefor sample breakthrough during loading, and efficient use of wash andelution fluids.

FIG. 2 is an illustration of a single well embodiment of the presentinvention where the internal tapered well geometry is segmented,providing an exit spout (17) downstream from the smaller second porousfilter (13), a lower tapered section (18) containing both the smallerporous filter (13) and a bed of sorbent particles (12), an upper taperedsection (19) which contains the larger first porous filter (14) upstreamfrom the sorbent bed (12), and a transition section (20) leading to anupper fluid reservoir (21) having a larger diameter opening (22).Segmentation of the internal taper in this way allows for SPE deviceswhich have larger capacity reservoirs while maintaining the advantagesof the present invention in a relatively short overall well height.

FIG. 3 is an illustration of a multi-well version of an SPE deviceincorporating the single well design of FIG. 2. Common multi-wellformats include plate designs based on the common 48, 96, and 384standard well formats.

The exit spout directs fluids into any suitable container. In thepreferred embodiment, shown in FIG. 5, the exit spout 17 geometry issubstantially trapezoidal. This geometry is used to prevent the exitingfluids from creeping up the exterior wall of the device and provideseffective beading and dropping of the exiting fluids. A semi-circularshape may also be used for the exit spout 17 as shown in FIG. 6.

The present invention can be used to purify samples prior to analysis,i.e., to isolate a desired target substance from an interferingsubstance in a sample medium, using a smaller elution volume thanheretofore possible with prior art SPE devices. Specifically, and in apreferred embodiment, the method of the present invention comprisesfirst conditioning the SPE device with any organic solvent that iscapable of wetting the surfaces of the device and sorbent particles.Illustrative examples of organic solvents that can be used in theconditioning step include, but are not limited to: polar or non-polarorganic solvents such as methanol and acetonitrile. The amount oforganic solvent used to condition the SPE device may vary and is notcritical to the present invention so long as the organic solvent is usedin an amount that wets the SPE device. Note that the solvent used inthis step of the inventive method also serves to remove contaminantsfrom the SPE device.

After the conditioning step, an aqueous solution is used to equilibratethe conditioned SPE device. The amount of aqueous solution used toequilibrate the SPE device may vary and is not critical to the presentinvention.

A prepared sample containing at least one target substance as well asinterfering components is then added to the SPE device usingconventional means that are well known to those skilled in the art. Theinventive method is not limited to a specific prepared sample or targetsubstance. For example, the prepared sample may be blood plasma, serum,urine, and other like samples that are capable of being purified bysolid phase extraction. Insofar as the target substance is concerned,the inventive SPE method works well on polar compounds, non-polarcompounds, acidic compounds, neutral compounds, basic compounds and anymixtures thereof.

Next, an aqueous solution is employed to remove the interferingsubstance from the SPE device and thereafter the target substance, whichis adsorbed onto the sorbent particles, is eluted from the SPE deviceusing an organic eluant that is capable of removing the adsorbed targetsubstances from the SPE device.

The following examples are given to illustrate the scope of the presentinvention. Because these examples are given for illustrative purposesonly, the present invention is not limited to the following examples.

EXAMPLES Example 1

A spherical porous polyethylene filter having a diameter of 0.075″ ispress sealed into a molded well cavity having a 5° included angle taperas shown in FIG. 2. A packed bed is formed within the 5° tapered wellusing 2 milligrams of Waters' Oasis® HLB, 30 micron sorbent particles. Aspherical porous polyethylene filter having a diameter of 0.100″ ispress sealed into the upper section of the well which contains a 1°included angle. The upper porous filter acts to both contain the sorbentparticles within the well, and to act as a sample pre-filter.

Example 2

A spherical porous polyethylene filter having a diameter of 0.058″ ispress sealed into a well having a 5° included angle taper as inEXAMPLE 1. A packed bed is formed within the 5° tapered well using 1milligram of Waters' Oasis® HLB brand, 30 micron sorbent particles. Aspherical porous polyethylene filter having a diameter of 0.100″ ispress sealed into the upper section of the well to both contain thesorbent particles within the well, and to act as a sample pre-filter.The resulting device contains one half the amount of sorbent as inEXAMPLE 1, but due to the smaller lower filter size, the bed ispositioned lower in the tapered well, with a bed shape that is wellsuited for effective performance.

Example 3

The SPE device of EXAMPLE 1 is placed on a vacuum manifold station witha collection vial positioned below the exit spout to collect fluidsexiting the device. A vacuum of 10″ Hg is applied to draw fluids throughthe device. The device is first conditioned by passing 100 μL methanolthrough the device, followed by 100 μL water. A spiked plasma sample isprepared by spiking 250 μL porcine plasma with 1.9 μg of amitriptyline,followed by dilution with 250 μL of 2% phosphoric acid in water. Thediluted spiked plasma sample is then drawn through the device. Afteraddition of the diluted, spiked plasma sample, the sorbent bed is washedusing 100 μL water. An elution step is then performed by passing 25 μLacetonitrile/methanol (80/20 by volume) through the sorbent bed, andcollecting into a clean collection vial. The resulting sample mixturecontains the target compound, free from plasma interferences,concentrated ten fold. The sample solution may be analyzed directly, orfurther evaporated and reconstituted in a solvent mixture suitable forthe intended analysis.

Example 4

The SPE device of EXAMPLE 1 is used in identical manner as described inExample 3, except that after eluting with 25 μL acetonitrile/methanol(80/20 by volume), an additional 25 μL water is drawn through thesorbent bed and into the same vial which contains the previously elutedsample compound. The resulting sample mixture contains the targetcompound, free from plasma interferences, concentrated five fold in a50% aqueous/organic solution, which is well suited for direct analysisusing HPLC.

Example 5

The model target compounds acetaminophen, N-acetyl-procainamide,betamethasone, caffeine, naproxen, amitriptyline, and propranolol wereobtained from Sigma Aldrich. The model target compound practolol waspurchased from Tocris. The Octadecyl (C₁₈) SD-C18 3M Empore™ HighPerformance Extraction Disk Plate (PN 6015) was purchased from FisherScientific. The Universal Resin (UR) 3M Empore™ High PerformanceExtraction Disk Plate (PN 6345) was purchased from VWR. The Ansys®Technologies, INC. Spec•C18 96-Well Plate (PN 596-03) was purchased fromAnsys Technologies, INC. The 5 mg Oasis® HLB Extraction Plate waspurchased from Waters (PN 186000309). A 2 mg amount of Oasis® HLB(Waters Corporation) was packed into a device similar to that shown inFIG. 1 with the sorbent contained between a lower polyethylene sphericalfrit of a diameter of 0.08″ at the outlet and an upper polyethylenespherical frit of a diameter of 0.1″ at the inlet. Organic solvents wereobtained from VWR (J. T. Baker HPLC grade).

Stock 1 mg/mL solutions of each of the following model target compoundswere made in 20/80 methanol/water (v/v): acetaminophen, practolol,N-acetyl procainamide, caffeine, propranolol, and amitriptyline. Stock 1mg/mL solutions of each of the following model target compounds weremade in 80/20 methanol/water (v/v): naproxen, betamethasone, andibuprofen. The internal standard solution was prepared by adding equalparts of the ibuprofen stock solution to water (1:1). Appropriateamounts of the stock solutions were added to a pH 7 isotonic salinesolution to achieve the following concentration of model targetcompounds: Concentration Compound In Saline Test Mix practolol 5 μg/mLn-acetyl procainamide 7.5 μg/mL acetaminophen 5 μg/mL caffeine 7.5 μg/mLnaproxen 5 μg/mL Amitriptyline 7.5 μg/mL betamethasone 2.5 μg/mLpropranolol 40 μg/mL Phenyl acetic acid 150 μg/mLThe isotonic saline solution was prepared by adding 0.4 g KCl, 16.00 gNaCl, 0.4 g KH₂PO₄, and 2.3 g Na₂HPO₄ to 3 liters of water. The mixturewas allowed to dissolve completely before adjusting to pH 7 withconcentrated H₃PO₄.

All solid phase extraction devices were conditioned with 100 μL ofmethanol, followed by 100 μL of water. Care was taken not to allow thesorbent to dry out between the methanol and water rinse steps. 100 μL ofthe saline solution containing the target model compounds was drawnthrough the device typically using <4″ Hg vacuum. 100 μL of water wasdrawn through the device to wash the sorbent. 25 μL or 75 μL of an 80/20acetonitrile/methanol solution was drawn through the device to elute themodel target compounds. 50 μL of a 0.5 mg/mL ibuprofen internal standardsolution and an additional 25 μL of saline was added to each sampleprior to analysis.

Samples were analyzed by HPLC using the following gradient of 0.01%formic acid (D) to acetonitrile (C): Time Flow % A % B % C % D Curve2.00 0.0 0.0 0.0 100.0 7.33 2.00 0.0 0.0 65.0 35.0 6 8.60 3.00 0.0 0.0100.0 0.0 1 8.84 4.00 0.0 0.0 100.0 0.0 1 9.00 2.00 0.0 0.0 0.0 100.0 19.50 3.00 0.0 0.0 0.0 100.0 1 15.00 2.00 0.0 0.0 0.0 100.0 6 35.00 2.000.0 0.0 100.0 0.0 11 45.00 0.00 0.0 0.0 100.0 0.0 11

The column temperature was maintained at 30° C. using a Spark HollandMistral heater box. The HPLC system consisted of a Waters 600E SolventDelivery System, a Waters 717plus Autosampler, a Waters in-line vacuumdegasser, and a Waters 2487 Tunable UV detector set to 254 nm (samplingrate=2 points/sec). Millennium³² Chromatography Manager v3.20 was usedfor data acquisition and processing, and equipment control.

Separations were performed using a 3.5 μm Symmetry Shield RP8 4.6×75 mm(Waters part number Wat094263) column with a 5 μm Symmetry Shield RP8Sentry 3.9×20 mm guard column (Waters Part Number Wat200675). Theinjection volume was 10 μL for all standards, controls, and samples. Thetotal run time was 15 min.

The hold-up volume was determined for each of the devices tested. It wasdetermined by adding 50 μL or 75 μL, depending on estimates of thedevice's hold-up volume, of 50/50 isopropanol/water to 4 wells each. Thesolution was allowed to soak into the beds for 30 sec. A vacuum of first4″ Hg for 45 sec then 7″ Hg for 45 sec was used to draw the solutionthrough the devices and into total recovery vials (Waters PN186000837).The volume of solution in the vials was measured using an auto-pipette.The hold-up volume was determined by subtracting the collected volumefrom the added volume.

The recovery results in Table 1 show the performance difference betweenwhat is commercially available on the market today and this new tipdesign. The data shows that recoveries in 25 μL volumes ranged from 84%to 97% on the new tip device compared to 51%-86% on the best performingcommercially available device today, which also contains the samesorbent. This direct comparison illustrates how the new device formatimproves recoveries. Devices containing particles embedded in glassfibers or Teflon had recoveries that were substantially lower (0 to64%).

The target compounds are listed in Table 1 from the most polar at thetop of the list to the least polar. On the Oasis® HLB 5 mg 96-wellplate, the recovery results sharply decrease as the polarity of thecompounds decreases. The new tip device is able to give high recoveriesfor compounds having a wide range of chromatographic polarities. TABLE 1% Recoveries (n = 4) in 25 μL Elution Volumes with 80/20Acetonitrile/Methanol Inventive Device† Oasis ® Oasis ® HLB 5 mg Ansys ®Spec 3M Empore ™ HLB 2 mg Plate Plus C₁₈ Plate Universal Resin packedbed packed bed glass fiber disk teflon disk Elution Volume 25 μL 25 μL25 μL 25 μL N-acetyl 95.6 85.8 0 6.9 procainamide practolol 92.8 83.6 07.7 acetaminophen 93.1 77.5 50.4* 9.1 caffeine 97.0 82.3 41.3 9.8propranolol 89.3 70.2 0 1.8 amitriptyline 83.6 59.1 0 0 betamethasone91.5 59.9 24.3 0.7 naproxen 84.1 50.8 64.4 5.3 Max Recovery 97 86 64 10Min Recovery 84 51 0 0†Replicates of 3.*Breakthrough in the load and wash was 27%.

All others show <5% breakthrough.

The relative standard deviations (% RSDs) for the recoveries are shownin Table 2. They range from 0.9%-4% on the new tip design versus4.6%-10.5% on the best performing current state of the art device.Results with equivalent recoveries and reproducibilities to thoseobtained on the new tip design were not obtained on the existing 96-wellplates with less than 75 μL elutions. For all quantitative analyticalwork good reproducibility is essential and high recovery is desirable.For high sensitivity quantitative analytical work both are essential:good reproducibility and high recovery. TABLE 2 % RSDs (n = 4) forRecoveries in 25 μL Elution Volumes with 80/20 Acetonitrile/MethanolInventive Device† Oasis ® Oasis ® HLB 5 mg Ansys ® Spec 3M Empore ™ HLB2 mg Plate Plus C₁₈ Plate Universal Resin packed bed packed bed glassfiber disk teflon disk Elution Volume 25 μL 25 μL 25 μL 25 μL N-acetyl0.9 5.0 72.9 procainamide Practolol 1.2 6.1 69.1 Acetaminophen 1.6 6.410.0 59.2 Caffeine 1.7 4.6 8.6 58.0 Propranolol 1.6 6.7 71.3Amitriptyline 4.0 7.6 — Betamethasone 2.5 8.6 13.5 200.0 Naproxen 2.510.5 3.2 73.0 Max RSD 4.0 10.5 13.5 200.0 Min RSD 0.9 4.6 3.2 58.0†Replicates of 3.

Table 3 shows the recovery results obtained using a 75 μL elution volumeon commercially available 96 well SPE plates that have been specificallydesigned to minimize elution volume. The shortcoming of the Oasis HLB 5mg plate is that the recoveries vary with the polarity of the compoundsdue to insufficient elution volume. The shortcomings of the Ansys®device are two fold. First recoveries of the basic compounds areextremely poor due to secondary interactions with the sorbent and glassfiber. The addition of about 2% acetic acid or 2% ammonium hydroxide tothe elution solvent would improve recoveries. The manufacturer of thisdevice recommends using 500 μL or less to elute compounds from thisdevice.

Neutral model compounds like caffeine, a polar compound, andbetamethasone, a non-polar compound, do not suffer from this problem.The 78.9% recovery for caffeine, and 67.6% recovery for betamethasoneshow that 75 μL is not an adequate elution volume to recover a broadpolarity range of compounds from the Ansys plate.

The 3M Empore™ devices also show recovery problems for the bases. Inaddition, the 51% and 56% recoveries for betamethasone show that 75 μLelution volumes are not adequate to elute a broad polarity range ofcompounds from these devices. All four of these devices also suffer frombreakthrough of acetaminophen, a polar neutral compound. TABLE 3 %Recovery (n = 4) in 75 μL Elution Volumes with 80/20Acetonitrile/Methanol for Different 96-well Formats Ansys ® 3M 3MOasis ® Spec Plus Empore ™ Empore ™ HLB C18 plate Universal C18-SD plate5 mg glass fiber Resin plate plate packed bed disk Teflon disk Teflondisk Elution Volume 75 μL 75 μL 75 μL 75 μL N-acetyl 85.2 7.0 57.4 73.3procainamide Practolol 81.7 19.8 56.0 82.3** acetaminophen 86.3** 68.2*71.5** 76.6* Caffeine 93.1 78.9 75.1 92.9 Propranolol 83.8 1.8 33.5 41.5Amitriptyline 85.3 0.6 20.1 6.1 betamethasone 87.8 67.6 51.1 55.6Naproxen 79.9 85.0 62.8 70.8 Max Recovery 93.1 85.0 75.1 92.9 MinRecovery 79.9 0.6 20.1 6.1*Breakthrough in the load and wash was 19-20%.**Breakthrough in the load and wash was 5-8%.

All others show <5% breakthrough. TABLE 4 % RSDs for Recoveries (n = 4)in 75 μL Elution Volumes with 80/20 Acetonitrile/Methanol for Different96-well Formats Ansys ® 3M 3M Spec Plus Empore ™ Empore ™ Oasis ® C18plate Universal C18-SD HLB glass Resin plate plate plate 5 mg fiber diskteflon disk teflon disk Elution Volume 75 μL 75 μL 75 μL 75 μL N-acetyl9.7 21.3 9.1 5.5 procainamide Practolol 10.6 24.0 8.8 3.6 acetaminophen5.9 4.5 8.5 10.8 Caffeine 3.9 9.5 8.6 3.1 Propranolol 6.9 138.8 4.3 19.5Amitriptyline 3.8 200.0 13.8 47.5 betamethasone 1.3 16.7 5.5 35.0Naproxen 3.0 3.1 7.7 19.1 Max RSD 10.6 200.0 13.8 47.5 Min RSD 1.3 3.14.3 3.1

The hold-up volume for each of the devices tested was measured and isshown in Table 5 along with the recoveries for three model compounds.The recoveries for these model compounds are highest for the new tipdevice due to its low hold-up volume. The recoveries in Table 5 show atrend of lower recoveries for devices with higher hold-up volumes asillustrated in FIG. 4. In FIG. 4, % recovery in 25 μL is plotted againstthe device hold-up volumes (V) in μL. Diamonds indicate caffeine data,squares indicate betamethasone data and triangles indicate naproxen dataTABLE 5 The Effect of Hold-up Volume on Recovery in 25 μL ElutionVolumes Inventive 3M Empore ™ Device Oasis ® Oasis ® HLB 5 mg Ansys ®Spec Universal HLB 2 mg Plate Plus C₁₈ Plate Resisn Plate packed bedpacked bed glass fiber disk teflon disk Elution Volume 25.0 μL 25.0 μL25.0 μL 25.0 μL caffeine 97.0 82.3 41.3 9.8 betamethasone 91.5 59.9 24.30.7 naproxen 84.1 50.8 64.4 5.3 Device hold up 16.0 28.0 36.0 64.0volume (μL)

Packed beds having a bed height to top diameter ratio of <0.23 are notable to efficiently retain or elute compounds due to imperfections inthe packed bed. Simply going to a 2 mg amount of sorbent in the existingOasis® HLB plate will not provide a result comparable to those obtainedon the new device containing 2 mg. This is illustrated with the data inTable 6 showing lower recoveries for all but the most non-polarcompounds on the plate containing 2 mg of sorbent compared to the platecontaining 5 mg of sorbent. Table 7 shows that the RSDs are worse on the2 mg plate compared to the 5 mg plate. TABLE 6 Effect of Bed Height toTop Diameter Ratio on Recovery in 25 μL Inventive Oasis ® Oasis ® DeviceHLB 2 mg HLB 5 mg Oasis ® HLB 2 mg Plate Plate packed bed packed bedpacked bed Elution Volume 25 μL 25 μL 25 μL N-acetyl procainamide 95.653.3* 85.8 practolol 92.8 48.9* 83.6 acetaminophen 93.1 49.5* 77.5caffeine 97.0 59.4* 82.3 propranolol 89.3 62.2* 70.2 amitriptyline 83.660.3* 59.1 betamethasone 91.5 66.5* 59.9 naproxen 84.1 53.7* 50.8 Bedheight to top 0.97 0.092 0.23 diameter ratio*Breakthrough in the load and wash was 14-43%.

All others show <5% breakthrough. TABLE 7 Effect of Bed Height to TopDiameter on Recovery RSDs in 25 μL Inventive Device Oasis ® Oasis ®Oasis ® HLB 2 mg HLB 5 mg HLB 2 mg Plate Plate packed bed packed bedpacked bed Elution Volume 25 μL 25 μL 25 μL N-acetyl 0.9 44.6 5.0procainamide Practolol 1.2 46.5 6.1 acetaminophen 1.6 42.0 6.4 Caffeine1.7 38.0 4.6 propranolol 1.6 29.2 6.7 amitriptyline 4.0 22.7 7.6betamethasone 2.5 17.5 8.6 Naproxen 2.5 20.5 10.5 Bed height to top 0.970.092 0.23 diameter

Example 6

Devices similar to those shown in FIG. 1 were manually packed using1.0±0.05 mg of 30 μm Oasis® HLB (Waters Corporation) contained betweentwo polyethylene spherical frits: a 0.035″ spherical frit at the bottomof the bed and a 0.055″ spherical frit at the top of the bed. Sodiumchloride, Angiotensin II, and p-toluamide were obtained fromSigma-Aldrich. Triethylamine (TEA), glacial acetic acid, trifluoroaceticacid (TFA), and HPLC grade acetonitrile were obtained from J. T. Baker.The 15-mer oligodeoxythymidine (15-mer oligo T) was obtained fromMidland Certified Reagent Company (Midland Tex.). The 0.1 Mtriethylammonium acetate (TEAAc) was prepared by adding 2.21 mL ofglacial acid and 5.58 mL of triethylamine to 350 mL of H2O. The solutionwas mixed, adjusted to a volume of 400 mL and pH adjusted to pH 7 usingacetic acid. The 0.24% TFA, and 50% acetronitrile were prepared byvolume. The 50 mM NaCl was prepared by adding 0.0584 grams of NaCl to 1liter of H2O. The 0.1 M TEAAc with 50 mM NaCl was prepared by adding2.21 mL of glacial acid and 5.58 mL of triethylamine to 350 mL of 50 mMNaCl. The solution was mixed, adjusted to a volume of 400 mL with 50 mMNaCl and pH adjusted to pH 7 using acetic acid. The 60 μL DNA loadsample contained 1 μg of 15-mer oligo T and 1 μg of p-toluamide in the0.1 M TEAAc buffer with 50 mM NaCl. The 60 μL peptide load samplecontained 1 μg of Angiotensin II and 1 μg of p-toluamide in the 0.24%TFA. All solutions were drawn through the tips using a vacuum of <5″ Hg.

DNA Desalting Method:

1. Condition each tip (n=3) with 60 μL of acetonitrile followed by 60 μLof 0.1 M TEAAc buffer

2. Load 60 μL/tip of the DNA sample

3. Wash with 60 μL/tip of the 0.1 M TEAAc buffer followed by 60 μL/tipof H₂O

4. Elute each tip with 10 μL of 50% acetonitrile in H₂O

Peptide Method:

1. Condition each tip (n=4) with 60 μL of acetonitrile followed by 60 μLof 0.24% TFA

2. Load 60 μL/tip of the peptide sample

3. Wash with 20 μL of the 0.24% TFA followed by 20 μL of H₂O

4. Elute each tip with 10 μL of 50% acetonitrile in H₂O

The DNA desalting and peptide recovery results are presented in Table 8.The results in Table 8 show that excellent recoveries for smallmolecules (ie p-toluamide), biopolymers (15-mer oligo T) and peptidescan be obtained in 10 μL elution volumes. TABLE 8 Recoveries and RSDsfor 15-mer oligo T, Angiotensin II, and p-Toluamide % Recovery % RSD DNAMethod 15-mer oligo T 88.2 2.3 p-Toluamide 93.3 4.8 Peptide MethodAngiotensin II 101.6 1.2 p-Toluamide 96.7 4.7

According, it should be readily appreciated that the device and methodof the present invention has many practical applications. Additionally,although the preferred embodiments have been illustrated and described,it will be obvious to those skilled in the art that variousmodifications can be made without departing from the spirit and scope ofthis invention. Such modifications are to be considered as included inthe following claims.

1-2. (canceled)
 3. A method of separating a target substance frominterfering components in a sample medium comprising: providing an solidphase extraction device (SPE) comprising a reservoir with an opening forreceiving fluids; a well comprising an internally tapered wall, the wellhaving a wider interior diameter at a first end closest to the reservoirthan at a second end close to an exit spout, the well for conducting anextraction; an exit spout at the second end of the well for dischargingfluids; a first filter press sealed between the internally tapered wallsof the well for retaining insoluble components of the fluids; a secondfilter having a smaller diameter than the first filter press-sealedbetween the internally tapered walls of the well spaced apart and towardthe exit spout from the first filter; a quantity of sorbent particlespartial filing the volume in the well between the first and secondfilters; and a void volume between the quantity of sorbent particles andthe first filter for separating the quantity of sorbent particles fromthe first filter; and substantially isolating a target substance usingthe solid phase extraction device.
 4. The method according to claim 3wherein: the mass of sorbent particles is less than 5 milligrams.
 5. Themethod according to claim 3 wherein the isolating step comprises thesteps of: conditioning the SPE device with an organic solvent;equilibrating the SPE device with an aqueous solution; adding a preparedsample containing the target substances and interfering components tothe SPE device; washing the SPE device with an aqueous-organic solutionto remove interfering components; and eluting the adsorbed targetsubstances.
 6. The method according to claim 5 wherein: the targetsubstance is substantially eluted in less than 50 μL volume.
 7. Themethod according to claim 5 further comprising: diluting the elutedtarget substances by passing a diluent through the solid phaseextraction device.
 8. The method according to claim 3 where the samplemedium is blood plasma, urine or serum.
 9. The method according to ofclaim 3 where the target substance comprises at least one polarcompound, non-polar compound, acidic compound, neutral compound,biopolymer, basic compounds, and mixtures thereof.
 10. The method ofclaim 3 where greater than 80% of each absorbed target substances isisolated in at most 50 μL volume.
 11. The method according to claim 3wherein the sorbent particles comprise an ion exchange sorbent; areversed phase sorbent; or a normal phase sorbent.
 12. A method ofseparating a target substance from interfering components in a samplemedium as in claim 3 further comprising: the reservoir and well in amulti-well array. 13-18. (canceled)